Class B1 G protein-coupled receptors (GPCRs) constitute a distinct group of transmembrane domain proteins that are endogenously activated by medium-sized peptide hormones and control a wide range of important physiological functions. There is an unmet need to identify surrogate small molecule ligands that show intrinsic activity at these receptors. Such compounds could be used as pharmacological tools for defining binding pockets and the processes leading to receptor activation, with the ultimate goal of enabling the design of surrogate non-peptide modulators for therapeutic use. The difficulty in identifying such molecules in prior screening campaigns may indicate that potential leads that are present in available compound libraries are too weak for being detected by conventional approaches. The PI proposes to address this issue by using novel receptor constructs as screening tools that function as hypersentive sensors of intrinsic ligand activity. These constructs include (i) a constitutively active receptor mutant that amplifies partial agonist function as well as detects inverse agonists, and (ii) a receptor where the agonist moiety of GLP-1 is attached, leading to partial autoactivation and enhanced ligand detection. As Specific Aim 1, it is proposed to develop a high throughput screening assay, based on cAMP-induced luciferase readouts in a 384-well format. A three-pronged approach will be pursued that combines the strength of assessing compounds using the wild type GLP-1R (which has the highest signal-to-noise ratio for a full agonist) with the complementary advantages that are offered by the hypersensitive, constitutively active and autoactivated receptor variants. As Specific Aim 2, the applicants will perform a pilot small scale screen of biologically active compounds, as well as establish alternative functional readouts for validation and further assessment of apparent outliers. Once configured, the assay will be made available for screening a large and diverse non-peptide library through the Molecular Libraries Production Centers Network. These studies will explore a new screening paradigm that may not only advance the study of GLP-1R pharmacology, but may also provide an opportunity to overcome the current bottleneck in finding non-peptide ligands with intrinsic activity for other class B1 GPCRs. [unreadable] PUBLIC HEALTH RELEVANCE: Members of a distinct family of cell surface receptors regulate a wide array of important endocrine functions and provide promising therapeutic targets for the treatment of disease. However, efforts to better understand the structure-function relationships of these receptors, with the ultimate goal of facilitating the identification of drugs that can be applied orally, has been hampered by the fact that only very few small ligands as needed to conduct such studies are currently known. Using a receptor that is relevant for the treatment of diabetes as a model target, we propose to develop a novel screening method to search for cognate ligands with markedly higher sensitivity than can be achieved by conventional approaches. [unreadable] [unreadable] [unreadable]